As known there is an intense research and concentrated efforts towards the screening and discovery of new anti-cancer agents. This research relates to a wide range of malignancies, including central nervous system, colon, lung, melanoma, renal, ovarian and leukemia.
The clinical testing of primary tumor sensitivity and determination of drug dosage to be administered to individual cancer patients should maximize the elimination of malignant cells and would be useful in avoiding undesired toxic side effects exerted on healthy cells.
To-day, chemotherapy is still considered to have a main role in the treatment of a variety of human neoplasms. However,inherent and acquired anticancer drug resistance, are major obstacles towards a curative cancer chemotherapy. Thus, for example, the exposure of malignant cells or tumor cell lines to a single hydrophobic cytotoxic agent of natural origin, including: vinca alkaloids, anthracycline, epidophyllotoxins, actinomycin D, colchicine and taxol, frequently results in the emergence of cell populations exhibiting resistance to the selecting agent as well as to the remaining pleiotropic drugs that are structurally unrelated lipophilic antibiotics. This phenomenon, known as term multidrug resistance is gaining a great deal of interest. It appears, that this is a result of the overproduction of P-glycoprotein (P-170) which is an integral component of the mammalian plasma membrane. This membrane functions as an energy-dependent efflux transporter of some antitumor agents known as pleiotropic drugs. Increased P-170 expression has been detected in both normal tissues,including adrenal glands, kidney, lung, liver, colon and placenta as well as in a number of hematological malignancies including acute myeloid leukemia, multiple myeloma and non-Hodgkin's lymphoma and in untreated and treated breast cancer.
Taxol has shown very promising activity against ovarian cancer, advanced breast cancer, and other tumor types and has also been approved as an investigational drug by the F.D.A. However, the disadvantages as mentioned above for the cytotoxic agents, should be taken in account.
Several methods are used for the in-vitro assessment of antitumor drug sensitivity. These methods are based either on the colony-forming ability of cells (so called clonogenic) on cellular growth inhibition, or on cell proliferation. The widely used clonogenic cytotoxic assay was applied to non-adherent malignant cells by utilizing a soft agar-based colony formation technique. The clonogenicity-independent methods include a cell counting, after trypan blue exclusion, or using a radioactive nucleic acid precursor, for example, tritiated thymidine.
A microtiter assay based on cellular proliferation was specially developed for the assessment of anticancer drug sensitivity of mammalian cells that bear poor plating efficiencies. High-flux in-vitro antitumor drug screening methods that evaluate cell viability include the well-known colorimetric assay based on tetrazolium. According to this method, hydrogen acceptor reagent such as MTT [3,(4,5-dimethylthiazol-2-yl)] and its derivatives, when are present in viable cells become metabolically reduced, thus precipitating as insoluble coloured formazans, which are subsequently dissolved and determined by a spectrophotometer. However, clonogenic assays suffer from a number of disadvantages, the major one being the very low plating efficiency of tumor-derived cells as well as their tendency to form cell clumps. These limitations result in unreliable drug survival curves. Cellular viability assessed by colorimetric assays, such as the metabolic reduction of soluble tetrazolium salt derivatives to insoluble formazans, has several limitations including major variabilities in formazan formation between different cell lines, variabilities that result from cell culture age as well as from differences in glucose concentration at the time of assay, i.e. at the metabolic reduction of the tetrazolium salts. In order to obviate this disadvantage,it was suggested that an optimal tetrazolium concentration as well as an optimal incubation time, should be established for each cell line. However, this prior optimization of the assay conditions for each cell line, further complicates the existent terazolium-based technique.
A general disadvantage of all known methods for the in-vitro assesment of antitumor drug sensitivity, is the relatively long time involved in the obtaining some results,a factor which generally might be quite critical. The above brief review, illustrates some of the problems which exist for assays which are capable of evaluating in-vitro cell viability following exposure to anticancer drugs. Therefore, it is a long felt need for development of a reliable, rapid and versatile in-vitro assay for determining of cellular sensitivity and of multidrug resistance to antineoplastic agents in mammalian cells.
It is an object of the present invention to provide a simple method in-vitro for the identification and determination of multidrug resistance to various anticancer drugs in mammalian cells. It is another object of the present invention to provide a simple method in-vitro for the evaluating the effectiveness of certain chemotherapeutic regimens. It is yet another object of the present invention to provide a simple method in-vitro for the determination of multidrug resistance to various anti-cancer drugs in mammalian cells which is characterized by its reproducibility. It is a further object of the present invention to provide a simple method in-vitro for the determination of multidrug resistance to various anticancer drugs in mammalian cells which is characterized by its rapidity.